COVID-19 PCR: frequency of internal control inhibition in clinical practice

Introduction. Diagnosis of COVID-19 (coronavirus disease 2019) is best performed with real-time (quantitative) PCR (qPCR), the most sensitive method for detection and quantification of viral RNA. Using the Centers for Disease Control and Prevention (CDC) protocol, for each sample tested for the virus, three qPCR tests are performed, targeting the viral genes N1 and N2, in addition to the internal control gene RNase P. Samples in which internal control fails to amplify should be labelled ‘invalid’. Methods. This study aims to determine the frequency of inhibition of the RNase P gene used as an internal control in qPCR tests for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in a reference hospital in Southern Brazil during the COVID-19 pandemic (1 February 2021 to 31 March 2021). Results. A total 10, 311 samples were available for analysis. The mean cycle threshold (Ct) value for the RNAse P gene was 26.65 and the standard deviation was 3.18. A total of 252 samples were inhibited (2.4%) during the study period: amongst these, 77 (30.5%) showed late amplifications (beyond 2 standard deviations from the mean Ct value), and 175 (69.4%) revealed no fluorescence at all for the RNase P gene. Conclusions. This study showed a low percentage of inhibition using RNase P as an internal control in COVID-19 PCRs using the CDC protocol, thus proving the effectiveness of this protocol for identification of SARS-CoV-2 in clinical samples. Re-extraction was efficacious for samples that showed little or no fluorescence for the RNase P gene.


INTRODUCTION
In Brazil, coronavirus disease 2019 (COVID-19) disseminated quickly; only 18 months after the notification of its first reported occurrence in the country, more than 21 million cases have been reported, and more than 587000 patients have died of the disease [1]. Diagnosis of COVID-19 is best performed with real-time PCR (qPCR), the most sensitive method for detection and quantification of viral RNA [2]. The Centers for Disease Control and Prevention (CDC) established that for each sample tested for the virus, three qPCR tests should be performed, targeting the viral genes N1 and N2, in addition to the internal control gene RNase P [3]. For positivity, fluorescence curves should cross the cycle threshold (Ct) within the limit of 40 cycles. Failure to amplify the RNase P gene may indicate inadequate nucleic acid extraction, lack of sufficient human cells, inadequate setup and execution of the assay, or malfunction of the reagents/equipment. Samples in which internal control fails to amplify should be labelled 'invalid' , and in such cases a new nucleic acid extraction should be performed [3]. Searching the literature, it is surprising how little information is available on the frequency of internal control inhibition OPEN ACCESS in different centres. Thus, this study aims to determine the frequency of inhibition of the RNase P gene used as an internal control in qPCR tests for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in a reference hospital in Southern Brazil during the COVID-19 pandemic.

METHODS
All biological samples (mostly nasopharyngeal swab, sputum and liquor) tested for COVID-19 qPCR at the Molecular Biology Laboratory at Santa Casa de Misericordia de Porto Alegre, between 1 February 2021 and 31 March 2021, were considered for study. qPCR tests were performed according to the CDC protocol [3], in ABI 7500, StepOne or StepOne Plus thermocyclers. Samples were excluded if they were received outwith the predefined dates.
For nucleic acid extraction, three different methods were used: ReliaPrep Viral TNA Miniprep System (manual method using columns; Promega), Maxwell RSC Viral Total Nucleic Acid Purification Kit (semi-automated extraction based on magnetic beads; Promega) and Pure Link RNA Mini Kit (also a manual kit based on columns; Invitrogen).
To determine the frequency of internal control inhibition, we studied the Ct values of the RNAse P gene for each sample and compared that with the Ct value of the RNAse P gene for the whole population being tested. All samples amplifying with ≥2 standard deviations beyond the mean Ct value for the internal control gene were considered inhibited. We were also interested in studying the effect of re-amplification and/or re-extraction in the inhibited samples.
Descriptive statistics were used to summarize the data. The P values were considered statistically significant if ≤0.05. Data analysis was performed with SPSS 20.0 (IBM).

RESULTS
During the period of study, a total 11, 954 samples were processed in the laboratory, 4,164 were positive (34.8 %), 7,784 (65.1 %) were negative, and six were inconclusive or invalid (0.05 %). Among these, 10, 311 samples were available for analysis -13 % were excluded due to loss of data.
The samples were considered invalid when there were pre-analytical problems (i.e., when the collection tubes did not contain the swab), and inconclusive when internal control failed to amplify in the initial qPCR run. These samples were submitted to re-extraction and/or re-amplification.
The mean Ct value for the RNAse P gene was 26.65 and the standard deviation was 3.18. A total of 252 samples were inhibited (2.4 %) during the study period: amongst these, 77 (30.5 %) showed late amplifications (beyond 2 standard deviations from the mean Ct value), and 175 (69.4 %) revealed no fluorescence at all for the RNase P gene. Table 1, from the 175 samples that were totally inhibited for the RNAse P gene, 140 (80.0 %) were extracted with the ReliaPrep, 23 (13.1 %) with Maxwell and 12 (6.9 %) with Pure Link. Among those that showed late amplification for the RNAse P gene (n=77), 39 (50.6 %) were extracted with ReliaPrep and 38 (49.3 %) with Maxwell. Of the 252 inhibited samples, 68 were reported without any corrective action, 20 because these were positive samples for the novel coronavirus, 34 were negative but they showed late amplification, two were invalid and the other 12 were negative and did not undergo any measures of correction. On the other hand, 103 were submitted to nucleic acid re-extraction, resulting in 30 positive results for COVID-19 (29.1 %), 71 negative results (68.9 %) and two inconclusive results (1.9 %). Also, 81 samples were re-amplified (and not re-extracted), and eight of these were found to be positive for COVID-19 (9.9 %).

DISCUSSION
The RNase P gene is used in the CDC protocol as an internal control for detection of COVID-19, since this is a multi-copy gene that is abundant in the human genome, and therefore is easily detectable [4]. Despite the widespread use of qPCR tests in clinical medicine, it was surprising that at the time of writing of this manuscript there was a limited amount of data produced in the literature on the frequency of PCR inhibitors in clinical practice. Articles were found comparing pathogen-specific controls with human gene amplification [5][6][7], but none of them related to COVID-19.
[8] determined the degree and frequency of inhibition in qPCR for the detection of Pneumocystis jirovecii in respiratory samples. That differed from our work, which used all types of incoming biological samples and worked with SARS-CoV-2. In addition, the inhibition frequency in their study was 26.3 %, much higher than that in our study (2.4 %). Furthermore, the corrective measure for qPCR inhibition was dilution of the samples, whilst in our study samples were re-extracted or re-amplified. As can be seen from our results, the measures used in the laboratory were efficient, given that only two results were inconclusive and two invalid, totalling 1.58 % (4/252) of all samples that were found to be inhibited in the PCR test. The two invalid samples did not have swabs inside the tube used for sample collection, and therefore there was probably no DNA for analysis.
Moreover, the focus on minimizing the impact of false-negative results is increasing (i.e. assuming negative results truly represent the absence of qPCR diagnostic targets [9]), and therefore 103 samples were subjected to re-extraction and 128 to re-amplification. In addition, when the Maxwell method was used, the percentage of partially inhibited samples was higher than with the fully inhibited methods, showing that it has high recovery of the sample DNA.

CONCLUSIONS
This study showed a low percentage of inhibition using RNase P as an internal control in COVID-19 PCRs using the CDC protocol, particularly when the Maxwell system is used for nucleic acid extraction. Inhibitions can be promptly resolved by submitting samples to re-extraction, which suggests that inadequate mixing of sample with lysate buffer may account for most of the inhibited samples. We have therefore shown the effectiveness of this protocol for the identification of SARS-CoV-2 through the qPCR technique and of re-extracting and amplifying samples that showed little or no fluorescence for the RNase P gene.

Funding information
The authors received no specific grant from any funding agency.  Comments: This is a study that would be of interest to the field and community.

Author response to reviewers to Version 1
Porto Alegre, February 27 th 2023 Response to reviewer 3 1. Please provide the type of samples (e.g. nasopharyngeal swabs, sputum…etc), "mostly respiratory samples" is not clear. Also, sensitivities vary depending on whether the sample is acquired from the upper or lower RT. What is the status of these patients? Are these samples from screening efforts, or diagnosis after symptoms? What were the symptoms of the participants? Werethere an informed or implied consent provided for the participants? Thank you for your suggestions. Taking into account that we produced this paper with data that we already had from the PCR runs done in the laboratory daily routine, we do not have access to patient symptoms or diagnosis. Also, since none of their personal information, such as name, age, number/email for contact was used, when we submitted the paper idea to the ethic committee, they approved to be written without being necessary to have given a term of consent to the participants.

Was
The qPCR done in duplicates or triplicates? The PCR runs were done only once, except in cases of re-extraction and/or reamplification.
3. What was the reason behind using three extraction kits? With different samples? With varying number of samples? As we used existing data from the routine, the kits were used according to the laboratory's needs and availability for purchase in February and March 2021. 4. While using three extraction kits, each kit would provide different rate of sensitivity and specificity; how would you overcome the potential discrepancy of results? Since data on sensitivity and specificity of these kits were not available, we were not able to provide additional information on this.
5. Could you provide the name of software used for the statistical analysis? SPSS 20.0.
6. How much of the 10,311 samples were positive or negative? Unfortunately this data is not available.
7. On what basis the decision wasmade to re-extract or re-amplify the inhibited samples? 81 inhibited samples were not re-extracted, why? Samples were re-extracted when the amplification curve of a gene is very different from the pattern of the other, for example, in cases where we have a Ct of 28 in N1 and 40 in N2, or when there is amplification in only one of the genes. In all other situations, cases reamplification is usually chosen. As such, those 81 samples were not re-extracted because they were re-amplified.
8. "20 were released without any corrective action" does that mean they are positive or negative? Why are they still considered inhibited if no corrective action was conducted?
Doesn't it make more sense that a mistake happened during the first extraction or the amplification that resulted in the 'inhibited' status? How can you prove that the samples were actually inhibited? Duplicates? That decision to inform a final result without any corrective measure being taken was made by the lab technician in charge. This is real life data. But we agree that some of these samples might actually not been inhibited. 9. Why NCA was chosen for some samples, yet others were chosen for re-extraction or re-amplification? Have you tried similar measures with negative samples? As stated before, that decision to inform a final result without any corrective measure being taken was made by the lab technician in charge. No, we have not tried similar measures for negative samples.
10. From the numbers provided, Invitrogen kit is the only one showing 100% accuracy, wouldn't the conclusion be that this kit is the superior one rather than Maxwell?
Any statistical analysis to scientifically translate these numbers? Our data demonstrates that Maxwell was actually more efficient to remove inhibitors from clinical samples.
shouldn't you extract the same samples with the three kits? While using three extraction kits, each kit would provide different rate of sensitivity and specificity; how would you overcome the potential discrepancy of results? P3L33: Could you provide the name of software used for the statistical analysis? Results P3L38: How much of the 10,311 samples were positive or negative? P3L40: The standard deviation (3.18) reported is high, maybe you could adjust the parameters (e.g. excluding outliers). P4L48: Please mention the corrective action used P4L49-52: On what basis the decision was made to re-extract or re-amplify the inhibited samples? 81 inhibited samples were not re-extracted, why? What is the definition of inhibited and inconclusive result? "20 were released without any corrective action" does that mean they are positive or negative? Why are they still considered inhibited if no corrective action was conducted? The provided numbers don't add up to 252 Doesn't it make more sense that a mistake happened during the first extraction or the amplification that resulted in the 'inhibited' status? How can you prove that the samples were actually inhibited? Duplicates? Discussion P4L56-58: Maybe at the time of writing the manuscript this statement could be true, however, there are many in the literature nowadays. P4L61-66: what is the purpose of this paragraph? P5L81: 'that that' duplication error P5L83-84: Instead, one can argue that it has lower sensitivity than other kits. Also, it is scientifically incorrect to compare extraction kits without using the same samples on all of them, that is a massive compromise to the study design rigor and reproducibility. P7Table1: Why NCA was chosen for some samples, yet others were chosen for re-extraction or re-amplification? Have you tried similar measures with negative samples? What is the difference between invalid and inconclusive? From the numbers provided, Invitrogen kit is the only one showing 100% accuracy, wouldn't the conclusion be that this kit is the superior one rather than Maxwell? Any statistical analysis to scientifically translate these numbers? Comments: Through this writing, I inform you of the following observations, hoping they will be useful for the work that is being evaluated. The objectives of the methodology are established at the beginning and are met at the end of the study. Correct reference is made to the published literature. Although the methodology used is not described in detail, reference is made to it. The methods used have been correct according to the search needs in this study. They also establish the use of descriptive statistics for data management and it is reflected in the reported results. According to what has been reported, the ethical guidelines are followed as the research is approved by the hospital's ethics committee. The study is interesting, since it addresses a specific point of the CDC protocol, measuring the percentage of RNase P inhibition as an internal control in COVID-19 PCR reactions. Although it is known that the methodologies prior to their use were evaluated by parameters such as precision, reproducibility, detection limit, among other parameters for their validation. The importance of the study lies in the finding of the incidence in the inhibition, showing interesting data that indicate a low rate of inhibition using this methodology, and that in part was due